Mutations in the ?-cardiac myosin heavy chain are responsible for a large number of inherited Hypertrophic (HCM) and Dilated Cardiomyopathies (DCM). The objective of the proposal is the characterization of biophysical and biochemical properties of the mutant human cardiac myosin as an essential first step in identifying the changes in the structure and mechanism resulting in inherited cardiomyopathies. The difficulties in studying human cardiac myosin were due to the instability and heterogeneity of the protein obtained from patient tissues and the lack of an adequate expression system to produce high quality human ?-cardiac myosin in quantities that are sufficient for structural and kinetic analysis. We developed a mammalian expression system based on adenoviral infection of C2C12 cells enabling us to produce mg quantities of the human ? -cardiac motor domain and heavy meromyosin required for kinetic and structural studies. The crystal structure of the wild type ?-cardiac motor domain reveals a cluster of HCM and DCM mutations in a region linking the SH-1 helix, the converter domain, the wedge loop, and the relay helix/relay loop (Coupling Region) by a cluster of hydrophobic residues (Hydrophobic Nexus). The concerted movement of these elements is critical for mechanochemical coupling in the myosin motor. Docking and structural studies of the motor domain with the small molecule activator, omecamtiv mecarbil, indicate that it binds adjacent to the Hydrophobic Nexus and may act by influencing the mechanochemical coupling mechanism. Eight HCM/DCM mutants representing the benign and severe phenotypes residing in the Coupling Region were selected to study the changes in the structure and mechanism of the mutant motor by steady-state and rapid kinetics (Aim 1.) in vitro motility assays and single molecule force measurements (Aim 2.) and crystallographic structure (Aim 3). The effect of omecamtiv mecarbil on the coupling mechanism of mutant cardiac myosins may provide insights into the clinical management of the disease specifically for DCM patients. Understanding the molecular mechanism of cardiomyopathies will provide the basis for the development of new assays for targeted therapies.